High-throughput screening (HTS) methods for identifying antagonists of chemoattractant receptors often rely on detecting perturbations in downstream events, such as cell migration. In the case of chemokine receptors, leukocyte cell migration is often assayed. However, compounds disrupting cell membranes or blocking downstream events mimic these outcomes, masquerading as candidate antagonists. Considerable effort is then required to distinguish the genuine antagonists from those compounds or molecules that caused false positive signals. Identifying true antagonists, which represent only a very small fraction of the large collections of candidate antagonists analyzed in high-throughput screens, is a formidable task. Realizing any savings in time or expense can bring a new drug to patients more quickly and less expensively.
Conventional assays that are adapted for use in HTS methods for screening small molecule antagonists of ligand-receptor interactions and signaling are usually one-dimensional. That is, they isolate and assay only the ligand-receptor interaction or the cellular signaling that ligand binding initiates, but not both. Because of this separation of physical interaction (ligand-receptor binding) from function (receptor signaling and downstream events), false positive signals are often observed, slowing discovery and development. False positives are molecules that give the desired result for undesirable reasons; they are often seen in screens for small molecule antagonists. Small molecules that initially appear to be inhibitors of receptor-ligand binding interactions (a desired result) may give such a result, for example, either by inhibiting the receptor-ligand interaction by binding the target receptor or ligand (desirable reasons), or by sickening or killing cells, or wielding other undefined effects (undesirable reasons).
Furthermore, conventional drug discovery formats for chemoattractant receptor antagonists fail to identify all clinically important molecules, a consequence of false negative signals. False negatives mean that clinically important molecules are undetected and remain undiscovered. For example, a molecule that permits chemoattractant receptor ligand-chemoattractant receptor binding, but inhibits chemoattractant receptor signaling, will be hidden in an initial screen for inhibitors of ligand binding.
Chemoattractant molecules attract cells. For example, chemokines, a group of more than 40 small peptides (generally 7-10 kDa in size), act as molecular beacons for the recruitment, activation, and directed migration of T lymphocytes, neutrophils and macrophages of the immune system, flagging pathogens and tumor masses for destruction. While defending the individual from invading pathogens and tumors, the immune system can cause disease when improperly regulated. Chemokine-receptor binding is linked to G-protein-coupled signaling cascades to mediate chemoattractant and chemostimulant signaling functions.
Inappropriate chemokine signaling can either promote infections when not properly triggered (Forster et al., 1999) or lead to diseases associated with defective chemokine signaling, including asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, and atherosclerosis (reviewed in Rossi and Zlotnick, 2000). Because chemokines play pivotal roles in inflammation and lymphocyte development, the ability to specifically manipulate their activity will have enormous impact on ameliorating and halting diseases that currently have no satisfactory treatment. Chemokine receptor antagonists can be used to obviate the generalized and complicating effects of costly immunosuppressive pharmaceuticals in transplant rejection (reviewed in DeVries et al., 1999).
To expedite the identification of chemoattractant receptor antagonists, such as those for chemokine receptors, an assay that weeds out false signals by testing both chemoattractant receptor binding and a biological function would hasten drug development.